Retinoic acid (RA) and its analogs (retinoids) exert diverse biological effects on normal growth, fetal development, cell differentiation, morphogenesis, metabolism and homeostasis (Deluca, 1991, FASEB J. 5:2924-2933; Sporn et al., 1994, The Retinoids: Biology, Chemistry and Medicine, 2nd Ed., Raven Press, New York). Retinoids have been linked to the induction of apoptosis (Ahuja et al., 1997, Dev. Biol. 208:466-481). Effects of retinoids have been shown to be mediated through two classes of nuclear receptors, i.e., the retinoic acid receptors (RARs) and the retinoid X receptors, which are members of the steroid/thyroid hormone receptor superfamily (Pfahl, 1994, Semin. Cell. Biol. 5:95-103; Mangelsdorf et al., 1995, Cell 83:841-850; Chambon, 1996, FASEB J. 10:940-954).
The mannose 6-phosphate/insulin-like growth factor II receptor (M6P/IGF2R protein) is a multifunctional transmembrane glycoprotein that consists of a 300 kDa single polypeptide chain with a large extracellular domain containing 15 repeat regions and a small cytoplasmic domain (Kornfeld, 1992, Ann. Rev. Biochem. 61:307-330). The expression of this receptor is developmentally regulated, with the receptor being high in fetal and neonatal tissues (including plasma and heart) and declining postnatally (Nissley et al., 1993, Mol. Reprod. Dev. 35:408-413). This receptor binds both mannose 6-phosphate (M6P) and insulin-like growth factor II (IGF-II) at separate sites (MacDonal et al., 1988, Science 239:1134-1137; Morgan et al., 1987, Nature 329:301-307; Kiess et al., 1988, J. Biol. Chem. 263:9339-9344). One function of the receptor is to bind and transport M6P-bearing glycoproteins (e.g. lysosomal enzymes) from the trans-Golgi network or the cell surface to lysosomes (Kornfeld, supra). The cell surface M6P/IGF-II receptor also binds and internalizes IGF-II, resulting in the lysosomal degradation of this ligand (Oka et al., J.Biol. Chem. 260:9435-9442). Thus, the receptor suppresses IGF-II proliferative effects. In addition, the M6P/IGF2R protein binds the latent transforming growth factor-xcex2 (LTGF-xcex2), permitting cleavage into its active form (TGF-xcex2), a potent growth inhibitor for most cell types (Dennis, et al., 1991, Proc. Natl. Acad. Sci. USA 88:580-584). Thus, the M6P/IGF-II receptor plays a role in the regulation of cell growth, and it has anti-cancer activity (Hankins et al., Oncogene 12:2003-2009; Sousa et al., 1995, Nature Genetics 11:447-449; Wang et al., 1997, Cancer Res. 57:2543-2546).
We have unexpectedly discovered that RA binds to the M6P/IGF2R protein with high affinity and thereby enhances the biological functions of the receptor. Those functions include the binding of M6P-ligands, the trafficking of lysosomal enzymes from the TGN to the lysosomes, and internalization (endocytosis) of exogenous M6P-containing glycoproteins and IGF-II. The interaction of RA with the M6P/IGF2R protein inhibits cell proliferation or induces apoptosis, or both.
Based on these discoveries, the invention provides a method for inhibiting proliferation of a cell or inducing apoptosis. In general, the method includes the steps of providing an elevated level of M6P/IGF2R protein in the cell; and contacting the cell with a compound that binds to the M6P/IGF2R protein. The elevated level of M6P/IGF2R protein in the cell can be achieved by providing a nucleic acid vector containing a M6P/IGF2R-encoding nucleotide sequence operably linked to an expression control sequence, and introducing the vector into the cell, where the M6P/IGF2R-encoding nucleotide sequence expresses M6P/IGF2R protein. The method can be used to inhibit proliferation of a cancer cell or induce apoptosis in a cancer cell. The cancer cell can be in vivo, e.g., in a human or a nonhuman mammal.
In preferred embodiments of the invention, the compound that binds to the M6P/IGF2R protein is a retinoid, but the compound is not required to be a retinoid. Examples of retinoids useful in the invention include retinoic acid, 13-cis-retinoic acid, 9-cis-retinoic acid, retinol, and retinol acetate.
The invention also provides another method for inhibiting proliferation of a cell or inducing apoptosis. This method includes contacting the cell with a retinoid that binds to an M6P/IGF2R protein and does not substantially affect a nuclear retinoic acid receptor-mediated process. Optionally, this method includes providing an elevated level of M6P/IGF2R protein in the cell (as described above).
The invention also provides a method for potentiating an MGP/IGF2R-mediated effect in a cell. The method includes contacting the cell with a first compound that binds directly to a first binding site on an M6P/IGF2R protein, and contacting the cell with a second compound that binds to a second binding site on an M6P/IGF2R protein. The first compound can be mannose-6-phosphate or an insulin-like growth factor. In some embodiments, the second compound does not substantially affect a nuclear retinoic acid receptor-mediated process. Preferably, the second compound is a retinoid, e.g., retinoic acid, 13-cis-retindic acid, 9-cis-retinoic acid, retinol, or retinol acetate. In preferred embodiments, the second binding site is located in repeat 15 of the M6P/IGF2R protein, and it includes amino acids 2213 to 2258 of the M6P/IGF2R protein. The method can be used in a cancer cell. The cancer cell can be in vivo, e.g., in a human or a nonhuman mammal. Typically, the M6P/IGF2R-mediated effect is inhibition of cell proliferation or induction of apoptosis.
The invention also provides an in vitro screening method for identification of compounds that bind directly to the retinoic acid binding site of a M6P/IGF2R protein. The method includes providing M6P/IGFR2 protein substantially free of nuclear retinoic acid receptor proteins; contacting the M6P/IGFR2 protein with a candidate compound; and detecting binding of the candidate compound to the M6P/IGFR2 protein. Preferably, the M6P/IGFR2 protein is immobilized, for example on an affinity chromatography column. Preferably, the candidate compound is a retinoid. The binding can be detected by a radioactive label. Preferred binding assays involve competitive inhibition.
The invention also provides a cellular screening method for identification of compounds that bind directly to the retinoic acid binding site of a M6P/IGF2R protein. The method includes providing a test cell; contacting the test cell with a candidate compound; and detecting enhancement of an M6P/IGF2R protein-related process in the cell. Examples of M6P/IGF2R protein-related processes are M6P/IGF2R protein trafficking in the cell, and an increase in an M6P/IGF2R protein-mediated function. Examples of M6P/IGF2R protein-mediated functions are binding of M6P/IGF2R protein to mannose-6-phosphate, and endocytosis of lysosomal proteins.
As used herein, xe2x80x9cretinoic acidxe2x80x9d or xe2x80x9cRAxe2x80x9d means all-trans-retinoic acid, unless otherwise indicated.
As used herein, xe2x80x9cretinoidxe2x80x9d includes retinoic acid, unless otherwise indicated.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present application, including definitions will control. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference.
Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described below. The materials, methods, and examples are illustrative only and not intended to be limiting. Other features and advantages of the invention will be apparent from the detailed description, and from the claims.